Establishment of the non-native horned-face bee Osmia cornifrons and the taurus mason bee Osmia taurus (Hymenoptera: Megachilidae) in Canada

Established populations of the non-native horned-face bee, Osmia cornifrons (Radoszkowski, 1887), and the taurus mason bee, Osmia taurus Smith, 1873 (Hymenoptera: Megachilidae), have been identified from Canada for the first time. In the US, the importation of O. cornifrons, beginning in the 1970s, led to its release for agricultural crop pollination and spread across the country. In this article, we report on O. cornifrons captured while sampling wild bees in Toronto, Ontario using hand nets, bug vacuums, and vane traps, as well as established populations in trap nests, from 2017–2020. The morphologically similar O. taurus, which was accidentally introduced to the US with shipments of imported O. cornifrons, was also recorded in our samples. Recently, a few individual O. taurus specimens have been identified from Ontario and Quebec; however, the extent of our sampling included nests, indicating it is also established in Canada. Others have shown its population growth to have been associated with concordant declines in abundances of native mason bee species in the US, and similar impacts are possible in Canada if action is not taken. We propose three non-mutually exclusive possible pathways for the arrival of O. cornifrons, as well as O. taurus, in Canada: (1) natural migration northward from non-native populations in the US, (2) international importation in the 1980s–2000s to support agricultural research programs, and (3) unintentional release of mason bee cocoons purchased from non-local vendors. We argue that a focus on enhancing populations of locally occurring native bees and stronger policy on the importation and sale of non-native bees are needed.


INTRODUCTION
Mason bees in the genus Osmia Panzer, 1806 (Hymenoptera: Megachilidae) are among the most important solitary bees used in agricultural systems. In particular, the horned-face bee, Osmia cornifrons (Radoszkowski, 1887) is valued for pollination of spring-flowering Megachilidae) promote the spread of the invasive yellow star-thistle, Centaurea solstitialis L. (Asterales: Asteraceae), in California (Barthell et al., 2001). Thornton (2004) found that non-native Megachile sculpturalis Smith, 1853 was a frequent visitor to the invasive kudzu, Pueraria DC. (Fabales: Fabaceae), in North Carolina. Non-native bees may also bring with them pests and diseases from their home range that can spread to native species. For example, Hedtke et al. (2015) demonstrated that O. cornifrons populations in New York were host to chalkbrood (Ascosphaera L. S. Olive & Spiltoir (Ascosphaerales: Ascosphaeraceae)), including an associated species from Japan, Ascosphaera naganensis Skou, 1988. This pathogen may have spread into populations of the native mason bee Osmia lignaria Say, 1837 and be implicated in its recent decline (LeCroy et al., 2020). On the other hand, some non-native cavity-nesting bees may have minimal impact on native bee communities. Megachile rotundata (Fabricius, 1787), for instance, has been present in North America since the 1940s and is now widespread, but there is no definitive evidence of it competitively excluding native species of Megachile Latreille, 1802 (Pitts-Singer & Cane, 2011).
In this article, we document for the first time the identification and establishment of O. cornifrons in Canada following observational reporting from surveys associated with separate studies using three sampling methods from 2017-2020. We also report an increase in the number of individual specimens of the morphologically similar O. taurus collected in Canada. For both species, we present evidence of established populations after uncovering nests from which we reared adults and report on fecundity, parasitism, and mortality. We suggest three non-mutually exclusive possible pathways for the arrival of O. cornifrons and expansion of O. taurus in Canada. These include (1) natural migration northward from non-native populations in the US, (2) international importation in the 1980s-2000s to support agricultural research programs, and (3) unintentional release of mason bee cocoons purchased from non-local vendors.

METHODS
Sampling for wild bees in spring occurred across Toronto, Ontario, Canada to answer various ecological questions that are not described here. Sampling was conducted using three primary methods. The first involved the use of trap nests over 6 years spread over two time periods: 2011-2013 and 2018-2020. Trap nests were made of cardboard tubes of three different inner diameter sizes-3.4, 5.5, and 7.6 mm-encased in PVC pipes (see MacIvor & Packer, 2015 for additional details on design). The second was by targeted surveys in 2017 and 2019 on flowers of eastern redbud, Cercis canadensis L. (Fabales: Fabaceae), using hand nets and bee vacuums (2820GA Heavy Duty Hand-held Vac-Aspirator; BioQuip, Rancho Dominguez, CA, USA). The third included the use of blue vane traps (SpringStar Inc., Woodinville, WA, USA) suspended on the branches of eastern redbud trees, before, during, and after redbud bloom in 2018. All sampling occurred in Toronto, the largest and most populated city in Canada, in private yards and seminatural public parks that contained a mixture of meadow and woodland vegetation, all within or surrounded by a heavily urbanized landscape.
Osmia cornifrons is markedly distinct from the only two species of Osmia subgenus Osmia native to North America-O. lignaria Say, 1837 andO. ribifloris Cockerell, 1900. The females of the two native species have a black (as opposed to yellow) metasomal scopa and lack the distinct clypeal horn-like protrusions present in O. cornifrons (Rust, 1974). The males of O. lignaria and O. ribifloris have dark brown/black (as opposed to reddish orange) meso-and metatibial spurs; and their metasomal terga have mixed black and white (as opposed to entirely yellowish) pubescence (Rust, 1974). However, given the similarity of O. cornifrons to O. taurus, with which it has been confused, all sampled specimens were run through the key (and identified following the taxon concepts) of Yasumatsu & Hirashima (1950), with identifications of a subset of males confirmed by dissection and examination of the genitalia, which are markedly different between the two species. Digital focus-stacked images of exemplars were taken at the Canadian Museum of Nature (CMN) using the Leica Z16 APO A apochromatic zoom system combined with the Leica DFC495 and DMC5400 cameras and Leica Applications Suite. These were arranged into labeled figure plates in Adobe Photoshop 2020.
To confirm our identifications based on morphology, a subset of specimens was DNA barcoded. The DNA barcode region, a fragment of the cytochrome c oxidase subunit I (COI) mitochondrial gene Hebert, Ratnasingham & de Waard, 2003), was amplified from template DNA and sequenced at the CMN Laboratory of Molecular Biodiversity in Gatineau, Quebec. From each specimen, the right mid-and hind legs (and in one specimen also the left hind leg) were removed and ground in Bio Plas G-TubeÒ 2.0 mL Microcentrifuge Tubes containing~0.2 g of 1.0 mm silicon carbide sharp particles in a BeadBeater. DNA was then extracted from the ground tissues following a protocol adapted from Ivanova, de Waard & Hebert (2006).
The barcode region was amplified using the primers MLepF1 (Hajibabaei et al., 2006) and LepR1 (Hebert et al., 2004), which were also used for Sanger sequencing in both directions. The PCR reaction mixture (prepared in volumes of 15 or 50 mL) consisted of 7.05 or 29.5 mL ddH 2 O, 3 or 10 mL 5 × Q5Ò Reaction Buffer (New England Biolabs (NEB), Ipswich, MA, USA), 0.3 or 1 mL dNTPs (10 mM), 0.75 or 2.5 mL MLepF1 forward primer (10 mM), 0.75 or 2.5 mL LepR1 reverse primer (10 mM), 0.15 or 0.5 mL Q5Ò High-Fidelity DNA polymerase (0.3 U/rxn) (NEB), and 3 or 4 mL template DNA. For PCR, the following conditions were programed into an EppendorfÒ MastercyclerÒ Pro Thermal Cycler: 1 min at 94 C, five cycles of 40 s at 94 C, 40 s at 45 C, and 1 min at 72 C followed by 40 cycles of 40 s at 94 C, 40 s at 51 C, and 1 min at 72 C, and 5 min at 72 C.
The reaction mixture for sequencing consisted of 6.2 mL nuclease-free water, 1.8 mL 5 × ABI buffer, 0.5 mL primer, 0.5 mL BigDye, and 1 mL diluted PCR products for a total of 10 mL per sample. Thermocycling used the following reaction program: 30 cycles of 30 s at 96 C, 20 s at 50 C, and 4 min at 60 C. Sequencing products were transferred to a 96-well plastic plate and therein purified by adding 1 mL 125 mM EDTA, 1 mL 3M NaOAc, and 25 mL 99% ethanol to each sample (to precipitate the DNA) before being subjected to cold centrifugation, rinsing of the DNA pellet in 35 mL 70% ethanol, and additional cold centrifugation. The pellets were thoroughly dried before being resuspended in 15 mL Hi-Di Formamide. The sequencing reaction products were then denatured in a thermal cycler for 5 min at 95 C, cooled for 2 min, and loaded into an Applied BiosystemsÒ 3500xL Genetic Analyzer for genetic analysis.
ABI trace files were imported into Geneious ver. 11.1.5 (Kearse et al., 2012) for assembly into consensus sequences, each of which was assembled from two traces. In the same program, sequences were trimmed, checked for quality, and screened to make sure no stop codons were present in the correct reading frame before being uploaded to the Barcode of Life Data System (BOLD). All newly generated sequences were run through the BOLD Identification Engine (https://www.boldsystems.org/index.php/IDS_OpenIdEngine) for comparison to available barcode sequences for O. cornifrons in the BOLD reference library. Genetic distance was visually assessed in a neighbor-joining (NJ) tree generated on the BOLD website based on the Kimura 2 Parameter distance model (Kimura, 1980). All ten newly generated barcodes met the criteria to be assigned (in an automated process) a barcode index number (BIN), a unique operational taxonomic unit identifier that usually corresponds with real species limits (Ratnasingham & Hebert, 2007, 2013. Records for the barcoded specimens are maintained in the 'BGTA Bees of the Greater Toronto Area' project on BOLD and will be made public, with the sequences and metadata also released on GenBank (https://www.ncbi.nlm.nih.gov/genbank/), upon publication of this study.
In addition to barcoding select specimens of O. cornifrons, previously barcoded exemplars-a female (BOLD sample ID: BIOUG35836-H03) and male (BOLD sample ID: BIOUG05684-A02)-of O. taurus deposited in the collection of the Centre for Biodiversity Genomics (CBG) at the University of Guelph in Guelph, Ontario, were personally examined by TO and compared morphologically with specimens collected across Toronto. The female specimen was collected in Washington, D.C., USA in 2017 whereas the male was collected in Port Rowan, Ontario, in 2013.
To The first individual of O. cornifrons detected by our sampling was caught on May 24 th 2017 in Morningside Park in Toronto by CdK using a hand net (Fig. 5). The female bee was collected from a flowering eastern redbud tree, Cercis canadensis (Table 1). Five additional female O. cornifrons were subsequently collected between May 7 th and May 27 th 2018 from blue vane traps suspended from the branches of Cercis canadensis at two sites in Toronto. More efforts were directed with bee vacuums on C. canadensis trees in 2019, yielding 29 individual female O. cornifrons from five sites between May 23 rd and May 26 th (Fig. 5).
From trap nests, we did not record any O. cornifrons in the early years (2011-2013) of our investigations (see MacIvor & Packer, 2015). However, from trap nests set out in 2018 we recorded one nest at a single site from which two male adults emerged. In 2019, we recorded 38 brood from six nests at three sites (    observations). No restrictions were applied to these permits, and all were for commercial purposes. Fifteen of the permits were for importation to British Columbia, two went to Ontario, and one each to Alberta and New Brunswick.

DISCUSSION
In this article we document the occurrence of the horned-face mason bee, Osmia cornifrons, in Canada for the first time. This cavity-nesting bee is promoted as an effective pollinator in orchards and of other spring flowering crops. Its populations can grow quickly, and the life cycle includes a period of diapause, permitting its storage and transportation to new areas. As a result, the expansion of O. cornifrons is a good example of the managed-to-invasion continuum as documented in other bee species imported for agricultural purposes (Russo et al., 2021). We also record from our trap nest surveys in 2019 and 2020 the taurus mason bee, Osmia taurus, which has been detected in increasing numbers within Canada. Although no obvious impacts to native bees (e.g., Osmia spp.) were determinable from the occurrence records of O. cornifrons and O. taurus illustrated here alone, these patterns have been documented in the neighbouring US, which should be a cause for concern and call to action for Canadians wishing to preserve native bee diversity. In the following section we discuss possible pathways for how O. cornifrons, as well as O. taurus, arrived in Canada.   Paiero, 2022, personal communication). It is very possible that the species has migrated into Southern Ontario from populations in the eastern and Midwestern US, but more rigorous surveying in the southern regions of the province is needed. Encouraging people to post photos to iNaturalist for identification is one method for gathering evidence for potential pathways and to detect non-native species entering new regions or countries that might threaten native biodiversity (Mo & Mo, 2022). The Canadian records of O. cornifrons on GBIF that we were able to confirm from images show this species to be present along the Golden Horseshoe of Ontario, suggesting entry into Canada from the US via the Niagara Peninsula, which is technically an isthmus between Lake Erie and Lake Ontario. This is also suggested by the apparent lack of observations from areas further southwest in the province near the Michigan-Ontario border. This scenario seems particularly plausible in light of the fact that several non-managed non-native bee species-Hylaeus punctatus (Brullé, 1832) in the family Colletidae and Anthidium manicatum (Linnaeus, 1758), A. oblongatum (Illiger, 1806), Chelostoma rapunculi (Lepeletier, 1841), Hoplitis anthocopoides (Schenck, 1853), and Megachile sculpturalis in the family Megachilidae-were detected in Canada some years after their initial introduction into the eastern United States (Jaycox, 1967;Eickwort, 1970Eickwort, , 1980Snelling, 1983;Smith, 1991;Mangum & Brooks, 1997;Hoebeke & Wheeler, 1999;Mangum & Sumner, 2003;Romankova, 2003;Buck, Paiero & Marshall, 2005;Sheffield, Dumesh & Cheryomina, 2011). Only a few adventive species, including Hylaeus communis Nylander, 1852, H. pictipes Nylander, 1852 and Megachile ericetorum Lepeletier, 1841, were first detected in North America in eastern Canada and the Great Lakes region, having arrived there presumably via the St. Lawrence River, and these (apparently direct) introductions are Western Palaearctic in origin (Sheffield, Griswold & Richards, 2010;Gibbs & Dathe, 2017;Martins, Normandin & Ascher, 2017).

From former agricultural research-based releases in 1980s-2000s
It is illegal to import live bees from the US into Canada without a permit. To import live mason bees, the requirements are listed in Appendix 1, Section 6 of the CFIA policy D-12-02 (CFIA, 2022). A case-by-case evaluation would be made, based on species, origin, intended use, etc., to establish import requirements and/or any other safeguards that may Purchasing mason bee cocoons from online sellers in the US Immature cavity-nesting bees have a period of dormancy within cocoons, which allows vendors to mail them with minimal losses of the developing bees due to mortality. These are raised, collected, shipped, and sold to enhance pollination services, traditionally in an agricultural context. This market has recently turned to accommodate smaller numbers of shipped live bee cocoons for home gardeners wishing to supplement purchased bee hotels. However, bees purchased online could arrive from another part of the country (or a different country altogether) and include species different from the one (   could represent local releases of non-local bees, perhaps (and possibly, unwittingly) illegally obtained from US sources. In the US, where there is considerable trade in mason bee cocoons, it is illegal to export live bees to Canada without governmental permission. Federal law regulates the shipment of bees to Canada (7 CFR 322, Animal and Plant Health Inspection Service, 2004), but APHIS does not regulate interstate movement of bees within the US. The not-for-profit Orchard Bee Association (OBA) (http://www.orchardbee.org/) has best practices and shipping guidelines for US distributors and does not endorse the movement of mason bee cocoons from eastern to western states. This is because O. cornifrons and O. taurus are much more likely to be present in cocoons shipped from the eastern US and could impact western native bee populations. Despite these regulations and guidelines, the simplicity of shipping cocoons by mail necessitates more stringent requirements for those participating in the movement of these bees. This could begin with more resources allocated to public engagement to decrease online trade in cavity-nesting bees and increase gardening and other activities that enhance local, native bee populations. For example, Seekamp et al. (2016) showed that an engagement campaign by the U.S. Fish and Wildlife Service led to increased awareness among hobbyist aquarium owners in eight US states of the consequences of internet trade and the proper disposal of invasive species that could threaten the Laurentian Great Lakes region.

Next steps
The mode of entry to Canada for O. cornifrons and O. taurus was likely through natural migration across land borders of non-native populations in New York State and Michigan, but the unintentional release by Ontario residents purchasing mason bee cocoons online is also plausible. Further testing of genetic variation among specimens caught in Ontario could help to determine which of these two possible pathways was mainly if not entirely responsible for the establishment of these species within Canada. The promotion of wild bees (i.e., non-honeybees) as a means for people to engage in efforts to alleviate bee declines may result in misdirection of good intentions (MacIvor & Packer, 2015). The inadvertent support of wild bees using improperly evaluated methods such as purchasing mason bee cocoons online and from non-permitted and non-local sources must be highlighted as a potential growing concern for native wild bee conservation.
Given the declines in native mason bee species reported in the Mid-Atlantic US (LeCroy et al., 2020), it is imperative that Canadian officials be prepared for possible devastating impacts of increasing numbers of O. taurus as well as O. cornifrons on the Canadian native bee fauna in the future. The Orchard Bee Association in the US has developed protocols for detecting and managing the spread of mason bees in the country. A similar national association in Canada could play an important role in regulation and education around the movement of mason bee cocoons into different regions, provinces, and territories. At the provincial level, the Government of Ontario has been effective in limiting the spread of wood-boring beetles through public awareness campaigns and by restricting the movement of firewood (e.g., Asian long-horned beetle; CFIA, 2017). Similar methods could be employed to restrict mail delivery of mason bee cocoons. Even if marketed as native mason bee cocoons, the possibility of inadvertently including cocoons of O. cornifrons or O. taurus is not worth the risk of engaging in the practice of purchasing live bees from non-local sources.
Growing numbers of people are purchasing bee hotels to support native bee habitat, and some feel the need to supplement that purchase with live bee cocoons coming from unknown origins. We encourage agricultural suppliers and the public to avoid purchasing bee cocoons from out of province, and especially out of country, for release in conjunction with the placement of bee hotels. Instead, we recommend focusing on habitat stewardship and even the creation of nesting habitat (including bee hotels) to support local pollinators. This could also encourage trapping and raising native mason bee species and other cavity-nesting species for local sales and business development. Campaigns to educate the public on how to identify O. cornifrons and O. taurus and alert experts to verify identifications of suspected individuals are needed. This is increasingly possible through public data repositories such as iNaturalist, which are frequently checked and used in scientific research (Mesaglio & Callaghan, 2021;Howard et al., 2022). This will help to manage the spread of potentially invasive bee species and the associated consequences.
collections for representatives of O. cornifrons and O. taurus from Canada, especially Steve M. Paiero, Miriam Richards, and Nora Romero. Finally, we thank three anonymous reviewers for their constructive feedback on our submitted manuscript.

ADDITIONAL INFORMATION AND DECLARATIONS Funding
Funding support for Madison S Marshall and Charlotte W de Keyzer was provided by a Natural Science and Engineering Research Council of Canada (NSERC) discovery grant awarded to JSM (RGPIN-2018-05660). There was no additional external funding received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.